Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells

Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors’ identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions. Key features • Development of skeletal muscle organoid differentiation from human pluripotent stem cells, which recapitulates myogenesis. • Analysis of early embryonic and fetal myogenesis. • Provision of skeletal muscle progenitors for in vitro and in vivo analysis for up to 14 weeks of organoid culture. • In vitro myogenesis from patient-specific iPSCs allows to overcome the bottleneck of muscle biopsies of patients with pathological conditions.

Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis.Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies.3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid.Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors' identity and further production of organized structures of the neural plate margin and the dermomyotome.Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors.The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles.To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions.
Published: xxx xx, 2024 [15,16].The well-documented progression of diseases narrows the focus of the investigations to specific tissue and cell types.The prolongation of cultivation in vitro represents a challenge that overcomes embryonic gene expression and allows the observation of myogenesis until the onset of the disease in fetal or adult age.However, this appears mandatory to model genetic disorders like muscular dystrophies that have a relatively late onset.To overcome the lack of continued observation along myogenesis and disease onset, long-term culture approaches are essential to examine the mechanisms leading to muscle loss during myogenesis.We have established a 3D skeletal muscle organoid (SMO) system, which delivers a stable population of skeletal muscle progenitors to investigate human myogenesis during early embryonic and fetal development [17,18].Induction of paraxial mesoderm is mediated by BMP inhibition, Wnt activation, and bFGF application as in other 2D protocols [4].The application of retinoic acid, Shh, and WNT1A distinguishes this protocol from other 2D protocols and specifically leads to anterior somitic mesoderm and neural crest formation.The early Matrigel embedding is the decisive difference to all 2D protocols.In comparison to 2D protocols, our 3D protocol patterns Pax7-positive skeletal muscle progenitor cells with dormant, activated, and committed signatures of late fetal stages partially overlapping with adult satellite cell developmental scoring, which can be maintained for up to 14 weeks of culturing.Our 3D differentiation protocol does not go beyond 2D protocols to provide maturated physiologically responsive skeletal muscle cells, which we have demonstrated with the electrophysiological recording of organoid-derived cells of different origins [18].However, structural distinctions like the posterior paraxial mesoderm on day 5, specified neural crest dermomyotome on day 17, myogenic progenitor migration on day 23, and neural crest lineage arrest on day 35 cannot be similarly mimicked with PSC-differentiation in 2D protocols.Recently, three other groups have described protocols for skeletal muscle development within 3D organoid differentiation systems [19][20][21].In comparison to these protocols, we have focused on the myogenic progenitor cell identity in comparison to satellite cells by scRNAseq in greater detail (e.g., dormant, activated, and committed signatures) [18].We see the strength of our system as being able to retain Pax7-positive myogenic progenitors/satellite-like cells constantly even during longterm cultivation to study their alterations in muscular dystrophies when generated from patient-derived induced pluripotent stem cells (iPSCs).n/a 5 mL Poly(methacrylacid-2-hydroxyethylester) 120 mg/mL 1.2 g Total n/a 100 mL The coating solution is not sterilized after mixing, as the high ethanol content makes contaminations unlikely.After application to the culture plate, it is sterilized by UV treatment.Alternatively, the coating solution can be sterilized through a 0.25 μm filter.e. Count cells and adjust cell density to 200,000 cells/mL (=4,000 cells/20 μL).f.Use the lid of a 10 cm cell culture dish and place 20 μL drops onto it.Fill the dish with PBS for humidified condition and place the lid again on the dish.g.For EB formation, incubate the 10 cm cell culture dish overnight in the incubator at 37 °C and 5% CO2.Note: The following step takes place under a cell culture hood.

B. Generation of embryoid bodies (EBs) and differentiation of organoids
g. Wash EBs into a 10 cm cell culture dish using DMEM/F12.
Note: Embedded EB droplets appear reddish due to the Matrigel.Use fresh DMEM/F12 for better visualization when collecting them with a cut-off 1,000 μL pipette tip.The pipette tips are not coated before handling cells, EBs, or SMOs throughout the protocol.h.Prepare Di-CL media and place 1 mL in each low-attachment coated-plate well and warm it in the incubator.i. Use one cut-off 1,000 μL pipette tip to transfer embedded EBs into a low-attachment coated-plate well and incubate them at 37 °C and 5% CO2.j.Exchange media partly every day.
Note: Developing organoids are quite small.Control after media change if organoids are still present.
To avoid destroying organoids during media change, hold the culture plate at 45 degrees and carefully remove media by pointing at the top of the well and aspirating slowly.Organoids should never be dried out during media change.Important: Because myogenesis and migration take place within the Matrigel droplet, damaging the Matrigel during media change should be avoided at all times.
Note: This step requires extra caution to not lose or destroy the developing organoids, which can be challenging for beginners in all organoid protocols.The poor visibility of the embedded EB is a considerable criterion.A dark base under the cell culture plate helps to make it easier to recognize.Tilting the plate by just under 45° to the experimenter causes the organoids to shift to the lower edge Change media every second day.9. Day 15: Change media from Di-HF to DiX-H media by removing 75% and adding the same volume of media.
Change media every second day.10.From Day 30 on: Change media every third day.
Steps 5-10 are depicted in the protocol outline of Figures 1A and 3A.

Validation of protocol
The reproducibility of the protocol has been described within various paragraphs of the corresponding article Mavrommatis et al. (2023).The organoid approach was evaluated with six hiPSC lines with independent genetic backgrounds, with more than five independent derivations per line, for the control line (CB CD34+) with more than 20 derivations, always obtaining similar results.The organoids show very reproducible sizes during their development (Figure 1B of Mavrommatis et al. [18]).To further evaluate the reproducibility of organoid development, diffusion map analysis on qPCR-based expression analysis of 32 genes was applied at early stages, as well as integrative analysis on scRNAseq datasets of mature stages of organoid development from four independent iPSC lines.The data indicate highly conserved cluster representation of myogenic progenitors at all states, together with skeletal muscle myofibers, fibroadipogenic progenitors, and neural progenitors-related clusters (Figure 4, Supplemental Figure 6, Mavrommatis et al. [18]).

9 Published:
xxx xx, 2024 performing hanging drop method to generate EBs. 3. Day -1: Generation of EBs via hanging drop method: a. Dissolve 40 mg of PVA in 10 mL in TeSR TM -E8 TM /StemFlex TM , followed by sterilization via filtration (0.25 μM) and supplemented with 10 μM Y-27632 and 1% P/S.b.Discard media, rinse the dish with PBS to remove non-adherent cells, and incubate them with TrypLE for chemical dissociation for 3-5 min at 37 °C.c.Stop dissociation using basal media DMEM/F12.d.Spin the cells at 400 rpm for 5 min, discard the DMEM/F12, and resuspend in TeSR TM -E8 TM /StemFlex TM supplemented with 10 μM Y-27632 and 1% P/S and PVA.

4 .
Day 0: a. EBs should have a size of 200-250 μm.Note: If a cloud of dead single cells surrounds the EB, and the EB is below 200 μm in size, while repeating step B3, increase the concentration of Rock inhibitor to 1.5-3× to enhance cell survival.b.Thaw Matrigel on ice in the fridge; it should stay cold (<4 °C) to avoid premature polymerization throughout the process.c.Wash EBs into a dish using DMEM/F12.Note: The following step takes place under a stereoscope outside the hood after thorough disinfection of the used area.The application of 1.5× P/S in the medium prevents possible contaminations.d.Place individual EBs onto a Parafilm ® embedding surface using a 200 μL pipette.Remove excessive media but never leave EB without media (should remain approximately 5 μL of DMEM/F12).Wellformed EBs should not take any harm from it.e. Place a 30 μL Matrigel drop onto each EB, resuspend to ensure a homogeneous mix of EBs within the Matrigel droplet, and place the EB in the center of the drop using a 200 μL pipette.Note: To ensure homogeneous Matrigel polymerization: Resuspend 2-3× up and down and only place the EB within the Matrigel.Matrigel polymerizes faster due to the light source of the stereoscope.f.Incubate the embedded EBs in the incubator at 37 °C for 20-25 min.

7 .
Cite as: Kindler, U. et al. (2024).Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells.Bioprotocol 14(9): e4984.DOI: 10.21769/BioProtoc.4984.10 Published: xxx xx, 2024 and thus reduces the probability of damaging them. 5. Day 3: Change media from Di-CL to Di-CLF media by removing 75% and adding the same volume of media.6. Day 5: Change media from Di-CLF to Di-CLFR media by removing 75% and adding the same volume of media.Day 7: Change media from Di-CLFR to Di-LSW media by removing 75% and adding the same volume of media.Change media every second day.8. Day 11: Change media from Di-LSW to Di-HF media by removing 75% and adding the same volume of media.

Figure 1 .
Figure 1.Representative images of different stages of skeletal muscle organoid development.A. Brightfield images of myogenic development stages, with corresponding cytokines/growth factor applications.B. Representative immunocytochemistry pictures of mesodermal, neural, paraxial mesodermal, and neural crest origin during early stages.C. Representative immunocytochemistry pictures depict neural lineage arrest, myogenic progenitor migration, and skeletal muscle organoid formation at more mature stages following organoid culture progression.Dashed lines indicate the initial EB embedding site and growth before migration takes place.Scale bars: 500 μm in (C), 200 μm in A (Day 18-Day 60) and B (Day 17), 100 μm in A (Day -1-Day 17) and B (Day 5, Day 7, Day 11) (modified from Mavrommatis et al. [18]).

Figure 2 .
Figure 2. Representative single-cell RNA-seq profiling of late fetal myogenic progenitors at mature stages during skeletal muscle organoid development.A. t-SNE visualization of color-coded clustering (n = 4323 cells) at 12 weeks post differentiation highlights the predominant presence of skeletal muscle lineage, represented by clusters corresponding to myogenic progenitors (n = 1625 cells, 37% of total population) in non-dividing (n = 1317 cells) and mitotic (n = 308 cells) state, myoblasts (n = 731 cells), myocytes (n = 1147 cells), and myotubes (n = 442).Additionally, mesenchymal and neural lineages are represented by two smaller clusters of fibroadipogenic (n = 165 cells) and neural (n = 213 cells) progenitors, respectively.B. t-SNE plot visualization of color-coded clustering indicates myogenic progenitor subcluster with distinct molecular signatures: "dormant" PAX7 high /CHODL high /FBN1 high , "activated" CD44 high /CD98 + /MYOD1 + , and "mitotic" KI-67 + /CDK1+/TOP2A.C. Circle plot illustrates the aggregated cell-cell communication network for all clusters at week 12 of human skeletal muscle organoids development.Circle sizes are proportional to the number of cells in each cell group and edge width represents the communication probability.D. Pseudo-time ordering for myogenic progenitors and

Figure 3 .
Figure 3.In vivo potential of skeletal muscle organoids (SMO)-derived skeletal muscle progenitors.A. Illustration of the differentiation protocol including the timeline of culture media addition and factor incubation (C: CHIR99021, L: LDN193189, F: bFGF, H: HGF, R: retinoic acid, S: Sonic hedgehog, W: WNT1A).B. Brightfield microscopy images of iPSC-derived SMOs on the selected days of a 84-day culture period (scale bar: 100 μm).C. FACS of organoid-derived CD82-positive skeletal muscle progenitors and D. their evaluation 6 weeks after transplantation into the CTX-injured tibialis anterior of an immunodeficient mouse [staining with huLamin A/C (green), dystrophin (red), DAPI (blue); scale bar 50 μm].

. Sodium citrate solution (0.1 M) Reagent Final concentration Quantity or Volume
TeSR TM -E8 TM supplemented with 10 µM Y-27632 onto new Matrigel-coated 35 mm dishes.(Dissolve 0.5 mL of Matrigel in 24.5 mL of DMEM/F12.Add 2 mL of the solution to a 35 mm dish and polymerize the Matrigel at 37 °C for 2 h or overnight at room temperature (RT), followed by UV sterilization before use.)d.Culture the cells for two days and exchange TeSR TM -E8 TM daily.Use 2 mL of media for a 35 mm dish.

1 .
Day -4: Prepare low-attachment coated plates by adding 150 μL of coating solution into each 24-well plate well.Let the ethanol evaporate overnight and store plates at RT until use.2. Day -3/-2: Passage hiPSCs culture at 70%-80% confluency using the enzymatic dissociation approach to 1:3/1:4 ratio.The following day, refresh media without Rock inhibitor and, if: a. Culture is at 30%-40% confluence, proceed with generating EBs the following day.b.Culture is above 50% confluence, proceed with generating EBs the same day after refreshing the media without Rock inhibitor and culture the cells for at least 4-5 h before dissociating again for Cite as: Kindler, U. et al. (2024).Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells.Bioprotocol 14(9): e4984.DOI: 10.21769/BioProtoc.4984.